Cortical nitric oxide required for presynaptic long-term potentiation in the insular cortex

Nitric oxide (NO) is a key diffusible messenger in the mammalian brain. It has been proposed that NO may diffuse retrogradely into presynaptic terminals, contributing to the induction of hippocampal long-term potentiation (LTP). Here, we present novel evidence that NO is required for kainate receptor (KAR)-dependent presynaptic form of LTP (pre-LTP) in the adult insular cortex (IC). In the IC, we found that inhibition of NO synthase erased the maintenance of pre-LTP, while the induction of pre-LTP required the activation of KAR. Furthermore, NO is essential for pre-LTP induced between two pyramidal cells in the IC using the double patch-clamp recording. These results suggest that NO is required for homosynaptic pre-LTP in the IC. Our results present strong evidence for the critical roles of NO in pre-LTP in the IC. This article is part of a discussion meeting issue ‘Long-term potentiation: 50 years on’.


Introduction
Synaptic long-term potentiation (LTP) has been widely studied in the hippocampus, amygdala, cerebellum and many cerebral cortical areas, and it is considered to be the basic cellular model for learning and memory [1][2][3][4].Different forms of LTP have been reported in the hippocampus and cortex regarding basic mechanisms for the induction and expression of LTP [5].Two major forms of LTP have been reported in adult cortical synapses: N-methyld-aspartate receptor (NMDAR)-mediated postsynaptic LTP (post-LTP) and kainate receptor (KAR)-mediated presynaptic LTP (pre-LTP) [2,5,6].Similar forms of postsynaptic LTP induced by postsynaptic NMDAR have been reported in the hippocampus.In addition, there are reports of retrograde messengers, such as nitric oxide (NO), in the CA1 region of the hippocampus [7][8][9][10].In this case, the retrograde messengers generated by the activation of postsynaptic NMDARs are proposed to diffuse to presynaptic terminals to enhance the release of glutamate during LTP.
Among several candidates for retrograde messengers, NO receives the most attention [11].In the CA1 region of the hippocampus, it has been proposed that NO may be produced at postsynaptic sites and then diffuses to presynaptic terminals in which it activates the cyclic guanosine monophosphate-dependent signalling pathway [7][8][9][10].However, it has been reported that certain forms of CA1 LTP may be purely expressed by postsynaptic mechanisms, such as the modification of α-amino-3-hydroxy-5-methyl-isoxozole propionic acid receptor (AMPAR) or the insertion of newly synthesized AMPARs [12,13].Furthermore, a new form of KAR-dependent LTP in the hippocampus has been reported, which is purely expressed by the presynaptic mechanism [6].There is no report of the requirement of NO in this form of pre-LTP in the hippocampus.
The insular cortex (IC) is one of the critical regions for pain perception, taste aversion and emotional disorders [14][15][16][17].Immunohistochemical and autoradiographic results revealed that the IC, particularly the area surrounding the rhinal fissure, shows a markedly higher-level activity of NO synthase (NOS) and its associating coenzyme, nicotinamide adenine dinucleotide phosphate, in comparison with other cortical regions [18].However, little is known concerning the specific functions of NO in the IC.Here, we show that the novel presynaptic KAR-NO pathway contributes to pre-LTP in the IC.

Methods (a) Animals
Adult male C57BL/6 mice (8-12 weeks old) were used in most of the experiments.Mice were housed under a 12 L : 12 D cycle (onset at 7:00 a.m.) with food and water provided ad libitum.All mouse protocols were approved by the Animal Care and Use Committee of the University of Toronto (protocol ID, 20012315 and 20012148) and the Institutional Animal Care and Use Committee at Nihon University (ID, AP19DEN028-4).

(b) Slice preparation
The data reported below consist of results from 149 mice.We began preparing slices from a mouse that was kept in a bright room for approximately 2-3 h (9.00-10.00).Briefly, mice were deeply anaesthetized with isoflurane (5%).After decapitation, tissue blocks, including IC, were rapidly removed and stored for 2 min in ice-cold artificial cerebrospinal fluid (ACSF) containing the following (in mM): 124 NaCl, 2.5 KCl, 2 MgSO 4 , 1 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 and 10 d-glucose.Coronal slices were cut at a thickness of 300 µm using a microslicer (VT1200S, Leica, Wetzlar, Germany).Slices were incubated at room temperature for 1 h in a submersion-type holding chamber.ACSF was continuously aerated with a mixture of 95% O 2 /5% CO 2 .

(c) Whole-cell patch-clamp recording
The slices were placed in a recording chamber that was perfused continuously with normal ACSF at a rate of 0.8-1.0 ml min -1 .Whole-cell patch-clamp recordings were obtained from pyramidal cells in layer II/III using visualized observation with Nomarski optics (×40, Olympus BX51W1, Tokyo, Japan) and an infrared-sensitive video camera (OYL-150, Olympus).To obtain evoked excitatory postsynaptic current (eEPSC) responses, we delivered stimulations by a bipolar tungsten stimulating electrode (50 MΩ, WE3ST10.5F3;MicroProbes, Gaithersburg, MD, USA) placed in layer V of the IC.eEPSCs were induced by repetitive stimulations at 0.02 Hz to measure the baseline in the pre-LTP experiments.In our previous study in the IC of the rat, a laser scanning photo-stimulation technique revealed that pyramidal cells located in layers II/III are not likely to receive strong excitatory inputs from a region with a perpendicular angle to cortical face but rather two different regions, dorsal and ventral sites, with the oblique angles of layer V [19,20].Therefore, we placed the electrodes on the dorsal and ventral sites.To obtain unitary EPSC (uEPSC) responses, we carried out double whole-cell patch-clamp recording from adjacent pyramidal cells placed in layers II/III in the IC.Before uEPSC recordings, the voltage responses of pre-and post-synaptic cells were recorded by the injection of depolarizing current pulses (300 ms) to examine basic membrane properties, including repetitive firing patterns and frequency.Except for part of the experiments, presynaptic cells were recorded under current-clamp conditions during uEPSC recording to provide transmitter release from presynaptic cells.Short depolarizing current step pulses (1 ms, 2.5-4 nA) were applied to the presynaptic cells to induce action potentials.In the protocol for post-LTP and double patch-clamp recordings, eEPSCs and uEPSCs were delivered by repetitive stimulation at 0.033 Hz during the control and the period after the stimulus protocol.Induction of post-LTP and pre-LTP with double-patch recordings was performed within 10-15 min after establishing the whole-cell configuration to avoid the washout of intracellular contents that are critical for the establishment of synaptic plasticity.For the induction of pre-LTP of eEPSC, 240 paired presynaptic stimuli (with 50 ms inter-pulse intervals) were delivered at 2 Hz to the presynaptic fibres at a holding potential of −75 mV.To induce pre-and post-LTP in the IC, we used each protocol that was previously reported [6,19].For the induction of pre-LTP of uEPSC, 240 paired current injections to induce action potentials (2.5-4 nA, with 50 ms inter-pulse intervals) were delivered at 2 Hz to the presynaptic cells.For induction of post-LTP, 80 single stimuli were delivered at 2 Hz to the presynaptic fibres at a holding potential of 30 mV.The composition of the pipette solution was as follows (in mM): 145 K-gluconate, 5 mM NaCl, 1 mM MgCl 2 , 0.2 Ethylene glycolbis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2 Mg-Adenosine triphosphate (ATP) and 0.1 Na 3 -Guanosine-5'-triphosphate (GPT).The pipette solution had a pH of 7.3 and an osmolarity of 300 mOsm.Electrical signals were recorded with amplifiers (Multiclamp 700B, Molecular Devices, Sunnyvale, CA) and a digitizer (Digidata 1440A, Molecular Devices), observed online, and stored on a computer hard disk using Clampex (pClamp 10, Molecular Devices).For miniature EPSC (mEPSC) recordings, 50 µM D-AP5, 1 µM tetrodotoxin (TTX) and 100 µM picrotoxin were added to the perfusion solution.mEPSCs were detected at a threshold of three times the standard deviation (s.d.) of the baseline noise amplitude using event detection software.Access resistance was 15-30 MΩ and was monitored throughout the experiment.Data were discarded if access resistance changed >15% during an experiment.Data were filtered at 2 kHz and digitized at 10 kHz.

(e) Experimental design and analysis
Clampfit (pClamp 9, Molecular Devices) was used for the analysis of electrophysiological data.Averaged amplitude and paired-pulse ratio (PPR) determined by the ratio of the peak amplitude of the second eEPSCs to that of the first eEPSCs were obtained from 8 to 12 consecutive sweeps.The events in the mEPSC recordings were detected by WDETECTA, which was kindly provided by Professor John Huguenard (Stanford University); the number of events and their amplitudes were quantitatively analysed.The synaptic responses in the mEPSC and uEPSC recordings were detected at a threshold of three times s.d. of the baseline noise amplitude using event detection software.The failure of uEPSCs was defined to be less than three times the s.d. of the baseline.The values are expressed as the mean ± standard error of the mean (s.e.m.).The error bars in bar graphs represent s.e.m.Differences in the mean values between the two groups were compared with a paired t-test, Student's t-test.A Mann-Whitney U-test was employed for analysis of failure rate in uEPSCs.Differences with a probability (p) <0.05 were considered significant.

(a) Nitric oxide synthase inhibitor-sensitive long-term potentiation of excitatory synaptic responses in the insular cortex
In previous studies, we have demonstrated that the excitatory synapses in the IC showed post-LTP [15,19].Here, we like to focus on pre-LTP in the IC.First, we examined whether the excitatory synaptic response can be induced by stimulating layer V in the IC.Identification of the localization of excitatory sources in layer V projecting to layers II/III pyramidal cells was found (figure 1; see §2).Excitatory synaptic responses evoked by the electrodes placed in layer V were observed in layer II/III pyramidal cells which showed typical firing.
First, we investigated pre-LTP in the IC.After achieving a stable baseline recording in response to paired-pulse stimulation (inter-pulse interval of 50 ms) for at least 10 min, we applied the stimulus protocol for pre-LTP (2 Hz for 2 min) at a holding potential of −75 mV [6].We found that synaptic responses were significantly potentiated, and such potentiation persisted for at least 30-35 min (138.9± 6.9% of baseline; figure 1c,f).The PPR time course demonstrated that PPR changed during LTP (figure 1f lower).Second, we would like to know if pre-LTP in the IC may require NO.To clarify this possibility, we applied an NOS inhibitor, L-NAME (100 µM).As shown in figure 1d,g, interestingly, pre-LTP was completely blocked by the L-NAME application (95.8 ± 11.9% of baseline; figure 1g).Third, we applied an NO scavenger, cPTIO (300 µM), to block the NO pathway.There was little observation of the elevation of eEPSC amplitude during L-NAME after the pre-LTP protocol.Unlike the application of an NOS inhibitor, the increment of amplitude was induced initially after the stimulus protocol; however, the enhancement was abolished by applying cPTIO (110.1 ± 5.5% of baseline) approximately 20 min after the stimulation (figure 1h).Here, figure 1i shows the summarized results of normalized amplitude after 30 min of pre-LTP stimulation.There were significant differences in EPSCs between baseline and the period of 30-35 min after the stimulus protocol in the control.There were few differences in eEPSCs that applied L-NAME and cPTIO.These results demonstrated that the NO is necessary for pre-LTP induction in the IC.Significant changes in PPRs before and after LTP stimulation were shown in the control, but not L-NAME and cPTIO, suggesting that the LTP induced by pre-LTP stimulus protocol is mediated by pre-synaptic mechanisms (figure 1j).To examine whether NO is also required for post-LTP in the IC, we tested the effects of L-NAME on post-LTP stimulus protocol induced by the pairing protocol [6].We found that L-NAME has no effect on post-LTP (figure 2a).Although the remarkable elevation of eEPSC was observed 30-35 min after post-LTP stimulus protocol, there were no significant changes in PPRs after post-LTP induction in the IC (figure 2b,c).These data suggest that, unlike NO-mediated pre-LTP induction, the LTP induced by post-LTP stimulus protocol is mediated by postsynaptic mechanisms and is little affected by the NOS inhibitor.
LTP in hippocampal synapses [21].In the anterior cingulate cortex (ACC), Koga et al. reported that L-VDCCs also contribute to pre-LTP [6].Thus, we would like to test the possibility that pre-LTP in the IC can be blocked by the application of KAR and L-type VDCC antagonists.Figure 3a,b show the effect of the KAR antagonist before and after the stimulus protocol.UBP310 (10 µM) was added before the LTP-induced stimulus protocol blocked the induction of pre-LTP, but it had little effect on the maintenance of pre-LTP when the drug was added after the induction (figure 3d,e,h).Interestingly, the L-type VDCC blocker nifedipine (20 µM), which was applied before the application of the LTP-induction protocol, had little effect on pre-LTP (figure 3c,f,g).The results indicate that KARs play a selective role in the induction of pre-LTP, but L-type VDCCs are not involved in pre-LTP.

(c) Increasing nitric oxide production enhances presynaptic glutamate release
It has been reported that NO enhanced mEPSCs in hippocampal neurons [22].Since NO is essential for the induction of pre-LTP, we wanted to know if NO precursors may affect the frequency of mEPSCs, a measurement of presynaptic glutamate release [23].In the bath application of NO donner spermine NONOate (NONOate; 100-200 µM), the effect is selective on frequency and the amplitudes were not affected (figure 4a-c).We also repeated experiments with L-Arg, a substrate for NO synthase.Similarly, L-Arg also produced the same results.Furthermore, the effect of L-Arg was completely blocked by a NOS inhibitor L-NAME (100 µM).These data indicate that NO released by NO donner and L-Arg application affects presynaptic release machinery but does not affect the postsynaptic membrane figure 4. .Averaged events and amplitude of mEPSC during L-NAME (middle) and additional administration of L-Arg (right).There were no differences in frequency and amplitude of mEPSC (frequency, n =5, t (5) = 0.504, p = 0.636; amplitude, t (5) = 0.698, p = 0.516, paired t-test).

(d) Nitric oxide is required for presynaptic long-term potentiation between a pair of pyramidal cells in the insular cortex
It is presumed that employed electrical stimulus protocol, even minimal stimulation by an electrode, simultaneously activates various and unidentified fibres and presynaptic neurons projecting to postsynaptic neurons.Consequently, it is hardly possible to identify the origins of presynaptic fibres and neurons.The double-patch clamp technique is one of the best methods to determine the presynaptic neurons and enables us to record cortico-cortical-mediated EPSC directly.Therefore, we try to induce LTP-like responses in the cortico-cortical synaptic transmission in excitatory connections by the double-patch clamp technique.A sample digital image correlation (DIC) image of double patch pipettes shows that the connecting neuron pairs exist within approximately 20 µm (figure 5a).In this study, the connection rate in pairs between pyramidal cells was 9.3% (10 connections in 108 trials).LTP-like responses of uEPSC were induced by repetitive action potentials provided by depolarizing pulses to the presynaptic pyramidal cell.The plotted amplitude obtained from the same cell in figure 5b demonstrated a decrease in failure responses after the stimulus protocol (figure 5d). Figure 5f shows the summaries of pooled data and that there are significant differences in uEPSC amplitude, PPR and failure rate.On the other hand, L-NAME applied before the stimulus protocol suppressed the induction of LTP-like responses in uEPSCs (figure 5c).In the group that applied L-NAME, there were no significant changes in uEPSCs, PPR or failure rate (figure 5g).These results also suggest that LTP-like responses not only have NO-dependency but also are induced in the cortico-cortical synaptic transmission composed by a pair of pre-and post-synaptic IC pyramidal cells.

Figure 2 .
Figure 2. Little effect of NOS inhibitor on post-LTP induction.(a) Sample waveforms and time course plots of eEPSC amplitude with paired-pulse stimulation at 50 ms inter-stimulus interval in the baseline and after the stimulus protocol for induction post-LTP.L-NAME (100 µM) started to be applied before recording.(b) Time course plots of averaged plots of EPSCs (upper) and PPRs (lower) during baseline and after the stimulus protocol (EPSCs, n = 6, t 5 = 2.591, *p = 0.049, paired t-test).Note that the changes in EPSC kinetics were observed after the stimulus protocol for LTP induction.(c) Summary data of PPR in post-LTP (Post-LTP, n = 6, t 5 = 0.431, p = 0.685, paired t-test).

Figure 4 .
Figure 4. NO modulates release machinery in excitatory synapses and is provided by the presynaptic site.(a) and (b) Effects of NO donner and substrate for NO synthase on miniature EPSC (mEPSC).Sample traces showing mEPSC responses before (Control) and during application of spermine NONOate (NONOate, 100 µM left in (a) and L-arginine (L-Arg, 1 mM left in (b).Averaged inter-event intervals (IEI) and amplitude of mEPSC during application of NONOate (middle and right in (a) and L-Arg (middle and right in (b).There were significant differences in mEPSC events when comparing the control and after the application of NONOate (n = 12, t (11) = 2.381, *p = 0.036, paired t-test) and L-Arg (n = 8, t (7) = 2.667, *p = 0.032, paired t-test).There were little significant differences in mEPSC amplitude when comparing the control and after the application of NONOate (t (11) = 1.165, p = 0.268) and L-Arg (t (11) = 0.246, p = 0.813, paired t-test).(c) Suppressive effects of NOS inhibitor on L-Arg-induced enhancement of mEPSC events.Sample traces showing mEPSC responses during NOS inhibitor, L-NAME (100 µM), and additional application of L-Arg (1 mM left in (c)).Averaged events and amplitude of mEPSC during L-NAME (middle) and additional administration of L-Arg (right).There were no differences in frequency and amplitude of mEPSC (frequency, n =5, t (5) = 0.504, p = 0.636; amplitude, t (5) = 0.698, p = 0.516, paired t-test).

Figure 5 .
Figure 5. Pre-LTP in unitary EPSC (uEPSC) responses that were obtained from the excitatory connection between pyramidal cells.(a) Double whole-cell patch-clamp recordings were performed in layer II/III of IC under differential interference contrast infrared video microscopy (DIC, left).Experimental protocol of uEPSC events for induction of pre-LTP (right).(b) Pre-LTP-like responses appeared in the excitatory synaptic connection between Pyr1 and Pyr2.Firing properties of Pyr1 and Pyr2 were induced by depolarizing current pulse injections (300 ms) to the neurons shown in (a, left).Postsynaptic uEPSCs were recorded from Pyr2 before (the second trace from the bottom) and after (bottom trace) the stimulus protocol for pre-LTP.uEPSC responses were induced by paired depolarizing current pulses to Pyr1 (top traces) to induce action potentials (the second traces from the top).Grey and red waveforms indicate 11 consecutive traces and their averaged traces, respectively.Note that no failure responses were observed in pre-LTP-like responses after the stimulus protocol.(c) Pre-LTP-like responses were blocked by the NOS inhibitor.Firing properties of Pyr3 and Pyr4 (left).uEPSCs recorded from Pyr4 before and after the stimulus protocol to induce pre-LTP NOS inhibitor, L-NAME was applied before recording (right).Grey and blue waveforms indicate 11 consecutive traces and their averaged traces, respectively.(d) and (e) Time course plots of uEPSCs obtained from Pyr2 and Pyr4 showing in (b) and (c).(f) Summary of data showing uEPSC amplitude, PPR and failure rate (uEPSCs, n = 5, t 4 = 3.625, *p = 0.022; PPR, t 4 = 3.102, *p = 0.036, paired t-test; failure rate, †p = 0.043, Wilcoxon test).(g) Summary of data showing uEPSC amplitude, PPR and failure rate with the application of L-NAME (n = 5, t 4 = 0.117, p = 0.912; PPR, t 4 = 1.809, p = 0.145, paired t-test; failure rate, p = 0.109, Wilcoxon test).